The long-term goal of this research program is to understand the molecular mechanisms of human copper homeostasis in atomic detail. Copper serves as a cofactor for many key enzymes, but can also facilitate the formation of oxygen radicals, which can damage proteins, DNA, and lipids. The delivery of this essential yet potentially toxic metal ion to distinct cellular locations and particular target proteins is accomplished in part by metallochaperone proteins. Metallochaperones deliver copper ions to target proteins via specific protein-protein interactions. Three classes of eukaryotic copper chaperones have been identified: the Atx1-like chaperones, the copper chaperones for superoxide dismutase, and the copper chaperones for cytochrome c oxidase. The Atx1-like chaperones deliver copper to transport ATPases in the secretory pathway. These ATPases include the human Wilson and Menkes proteins, mutations in which lead to Wilson disease and Menkes syndrome, genetic disorders of copper metabolism. The copper chaperones for superoxide dismutase, known as the CCS proteins, provide copper,zinc superoxide dismutase (SOD1) with its catalytic copper ion. Mutations in SOD1 have been linked to familial amyotrophic lateral sclerosis (FALS). The copper chaperones for cytochrome c oxidase, Sco1 and Cox17, help assemble the CuA site in subunit 2 (Cox2). In humans, defects in cytochrome c oxidase lead to fatal cardioencephalomyopathy. The objective of the proposed work is to elucidate on the molecular level how proteins in these pathways bind copper, recognize and dock with physiological partners, and facilitate metal ion transfer. The metal binding properties of the copper chaperones and their target proteins will be studied by biophysical and crystallographic methods. Interactions between chaperones and target proteins will be investigated by biochemical, biophysical, and X-ray crystallographic techniques. In addition, interactions among the multiple metal binding domains of the transport ATPases will be examined. These approaches are expected to reveal molecular details of copper trafficking and provide new insight into the causes and treatments of diseases related to copper metabolism.